The gene Ras is mutated to remain in the active oncogenic state in one third of human cancers and as high as 90% in pancreatic cancer. We found that Ras oncogenesis is mediated differently between identical matched murine and human cells, with the latter depending upon the RalGEF pathway, suggesting a previously unappreciated key role for RalGEFs in Ras-mediated human cancer. RalGEFs activate two nearly identical G-proteins, RalA and RalB, although only the former fosters transformed and tumorigenic growth, due in part to the hypervariable C-terminus. We now propose two aims to further our understanding of the role of RalA in human cancer: Aim 1: Determine the function of the oncogenic C-terminus of RalA. Determining why RalA, but not RalB, can transform and promote tumor growth of human cells is important to understand Ras oncogenesis. Based on the fact that the C-terminus of RalA imparts oncogenic activity to RalB we will map the region responsible for this effect, identify the function of this region, and test if this function is required for tumorigenic growth of human pancreatic cancer cell lines. Aim 2: Elucidate the mechanism for RalA oncogenic signaling. How RalA is able to transform cells is unclear. We propose to attack this question at the two extreme ends of oncogenic Ral signaling. First, using loss-of-function analysis we will identify the effectors of RalA required for transformation, and test if the same effectors are also needed for tumorigenic growth of human pancreatic cancer cell lines. Second, as activation of RalA will eventually alter transcription of specific genes, we will identify a RalA oncogenic signature by comparing the transcription profile of identical cells in which RalA versus RalB is activated or repressed. The RalA oncogenic signature will be used to identify downstream pathways and specific genes associated with RalA oncogenesis. This signature will also be used to identify activation of RalA in cancers, ultimately for diagnosing cancer subtypes that rely on RalA. Completion of these aims at the basic cancer research level will better our understanding of RalA activation and signaling in cancer, and at the clinical level, provide a basis for targeting this in human cancers.